Use of IL-3RB to mediate antitumor effect of CAR-T cells through the JAK-STAT signaling pathway.

Abstract

2541 Background: The application of CAR-T in solid tumors has not yet demonstrated significant clinical benefits. The continuous proliferation and decreased terminal differentiation of CAR-T cells in vivo are considered potential strategies that could alter this predicament. Cytokines, as the 3rd signal for T-cell activation, play a crucial role in the lifecycle and fate of T cells. In this study, we innovatively incorporated the IL-3 receptor β chain (IL-3RB) into the CAR structure to enhance signal transduction, potentially activating the anti-tumor function of CAR-T and maintaining the persistent proliferative activity. Methods: Transcriptional profiling analysis performed on HER2 or VEGFR1 CAR-T cells, with a specific focus on the gp140 cytokine family (IL-3/IL-5/CSF-2) and the receptor expression, validated by ELISA and Western blot. By cloning CAR genes containing different structural domains of the IL-3RB into the lentiviral vector pWPXLd, CAR expression, activation, depletion, proliferation, cytotoxicity, pSTAT3 and pSTAT5, and cytokine release of CAR-T cells were assessed using flow cytometry (FCM), Western blot (WB), RTCA, CCK-8, and ELISA. Subcutaneous/abdominal injections of SKOV3 cells or SKOV3-Luc cells into NSG mice, followed treated by CAR-T cells. Tumor burden was regularly monitored, and FCM and Q-PCR were utilized to assess the quantity and phenotype of CAR-T cells in vivo. Results: RNA-sequence, ELISA and WB revealed that upon activation of CAR-T cells, the expression of genes such as IL-3/IL-5/CSF-2 rapidly increased. However, their receptors were hardly expressed. IL-3RB modification did not affect the phenotype of resting CAR-T cells. But, co-cultured with target cells, IL-3RB significantly enhanced the anti-tumor effects of CAR-T cells. This was manifested by more rapid activation, promotion of persistent proliferation, delayed exhaustion phenotype, secretion of higher levels of cytokines (IL-2, IFN-γ, TNF-α) and efficient cytotoxicity. IL-3RB-expressing CAR-T cells exhibited activation of the JAK kinase and STAT3, STAT5 transcription factor signaling pathways in vitro. We established solid tumor models of subcutaneous (SKOV3) and intraperitoneal metastasis, where HER2/VEGFR1 CAR-T cells partially inhibited tumor cell growth in vivo. In contrast, IL-3RB-enhanced CAR-T cells completely eradicated tumor cells, and even achieving a state of complete remission. FCM and Q-PCR also confirmed that IL-3RB significantly improved the proliferation and survival of CAR-T cells in vivo. Conclusions: IL-3RB is scarcely expressed in T cells and not regulated by T cell activation. IL-3RB-modified CAR-T cells exhibit superior persistence and significant anti-tumor advantages in a solid tumor through the JAK-STAT signaling pathway. Our innovative CAR structure design has the potential to demonstrate promising anti-tumor effects in clinical translational applications.